This invention relates to pairs of parent plants for producing hybrid seeds and to methods for producing plants with a desired phenotype. The desired phenotype is an active enzyme, a regulatory protein or a protein which affects the functionality and/or viability and/or structural integrity of a cell. Preferably, the desired phenotype is substantially absent from the parent plants/lines. In particular, the invention relates to parent plants and methods involving plant lines for producing male-sterile plants and seeds.
The present invention describes a protein complementation system, with a variety of different applications. The system can be explained and exemplified with reference to obtaining male-sterile plants and embryoless seeds although it is not limited to these applications.
The use of dominant Artificial Male Sterility (AMS) in plants is described in WO95/20668. This document describes a binary system using two genes which together (but not in isolation) cause male sterility. The genes are brought together by crossing plants, each parent being homozygous for the gene, which generates a homogenous population of male sterile plants. WO95/20668 describes several ways to implement the gene binary system, including the following:
i. a system based on activation of transcription: a transcriptionally inactive AMS gene is activated upon crossing by provision of the relevant transcription factor;
ii. a system based on activation of splicing: an AMS gene inactivated by the presence of an intron is activated upon crossing by provision of the relevant maturase;
iii. a system based on the suppression of a stop codon during translation: an AMS gene inactivated by introducing an artificial stop codon in the ORF, is activated upon crossing by provision of an artificial stop suppressor tRNA for the introduced stop codon.
iv. a system based on sequence-specific gene inactivation: One parent contains a modified male fertility gene and a transgene which inactivates only the unmodified male fertility gene. The other parent contains a transgene which inactivates only the modified male fertility gene. In the hybrid both the modified and unmodified male fertility genes are inactivated causing male sterility.
v. a system based on preventing restoration of male fertility by a restorer gene: the first parent contains the AMS gene and the restorer gene, and the second parent contains a gene inhibiting the action of the restorer gene.
However, the binary systems described above have so far proved complex to implement and have encountered a variety of difficulties.
For example, it has been found that the use of a suppressor tRNA (described in Betzner et al. 1996, Abstract of the 14th International Congress of Plant Reproduction, Lorne, Australia) can have deleterious consequences for some plant species. While this does not preclude its use, it does make the screening of suitable transgenic plants more labour intensive than desirable. Another example is the leakiness of the T7 promoter (described in EP-A-0589841). Some plants transformed with a T7 promoter driving Barnase were sterile in the absence of the T7 RNA polymerase. Again, this does not preclude use of the system but it does make it difficult to identify suitable transgenic plants. Furthermore, in certain plants the gene binary system is sub-optimal since not all of the required genetic elements are fully characterised.
Two areas of prior art have been explored which have resulted in a phenotype conferred to a plant by the combination of two proteins.
In 1989, Hiatt and coworkers (Nature, vol. 342, p. 76-78) described the production of a functional antibody in tobacco by crossing tobacco plants expressing a gamma immunoglobulin gene and a kappa immunoglobulin gene.
Problems were, however, encountered with this system. Since the light and heavy chains of an antibody interact through disulfide bridges, the bridges were unable to form in the reducing environment present in the cytoplasm. Assembly of a functional antibody in plants thus requires that both chains are targeted to the endoplasmic reticulum then secreted to the apoplast (the space between cells). The production of antibodies in plants has thus been limited to the production of secreted antibodies or the production of single chain antibodies.
In 1992 Lloyd et al. (Science, vol. 258, p. 1773-1775) described the transfer in Arabidopsis and tobacco of two maize genes coding for the transcription factors R and C1. Ectopic expression of these genes separately in heterologous plants has some effect on the transcription of endogenous genes. In particular the genes have some effect in isolation, and this may preclude their use for applied purposes. Co-expression of the two genes had more dramatic qualitative and quantitative effects, than expression of either gene alone. However, these genes have properties severely limiting their usefulness and their general inapplication is described in the paper.
It has been shown that the Arabidopsis transcription factors Apetala3 and Pistillata can be ectopically co-expressed, and jointly in concert cause a new phenotype in the Arabidopsis flower (Krizek and Meyerowitz, 1996, Development, vol. 122, p. 11-22). The limitations described above for the R/C1 proteins also apply in this case.
The present invention describes a protein complementation system which overcomes many of the problems and difficulties associated with known gene binary systems. The protein complementation system according to this invention is based on the expression of two or more gene sequences in a single plant, which polypeptides/proteins, associate, interact or come together to form an active enzyme, a regulatory protein or a protein which affects the functionality and/or viability and/or the structural integrity of a cell. Hereinafter, in this text all references to a protein which affects the structural integrity of a cell also describes a protein which may, in addition, or alternatively, affect the functionality and/or viability of a cell. Some polypeptides/proteins may fall in more than one of these categories None of the individual gene sequences present in a given plant lead to a significant phenotypic effect in these plants.
The present invention describes the creation of a plant which has a desired phenotype through expression of an active enzyme, regulatory protein or protein which affects the structural integrity or a cell (eg. a membrane destabilizing protein) The plant may be obtained by crossing a pair of parent plants a and b. Plant a contains one or more gene sequences which encode a polypeptide(s) or protein(s) (A) with little or no activity so that the desired phenotype is not significantly (or substantially) caused by expression of the one or more genes in plant a alone. Plant b also contains one or more gene sequences which encode a polypeptide(s) or protein(s) (B) also, with little or no activity so that the desired phenotype is not significantly (or substantially) caused by expression of the one or more genes in plant b alone. When plants a and b are crossed, the resulting hybrid expresses both polypeptides and/or proteins A and B. These two polypeptides/proteins associate, interact or come together to form an active enzyme, regulatory protein or protein which affects the structural integrity of the cell, with the result that the daughter plant displays the desired phenotype. NB: From hereon, when discussing the polypeptides/proteins A or B they will be referred to only as xe2x80x98polypeptidesxe2x80x99 for the sake of convenience.
This protein complementation binary system is simpler than the previously described binary systems since there is no need for interaction between genes, no required modification of the expression of genes and no modification of the level of expressed polypeptides in the daughter plant compared to the parent plants.